The use of biological markers which produce an optical signal has revolutionized several aspects of the life sciences and in particular has allowed for the real time observation of in vivo phenomenon such as endocytosis, embryonic development, infection, tumor development and calcium signaling. Increasingly these same technologies are also being used to visualize more and more biological and pharmaceutical processes in situ within a target cell, isolated tissue or whole organism.
There are essentially three mechanisms for generating a light emission based optical signal using a biological marker:
1. Bioluminescence.
2. Chemiluminescence.
3. Fluorescence.
Bioluminescence is the production and emission of light by a living organism or a fragment thereof such as an enzyme. Bioluminescence is a naturally occurring form of chemiluminescence (see below), where energy is released by a chemical reaction in the form of light emission. Adenosine triphosphate (ATP) is involved in most bioluminescent reactions and therefore generally bioluminescent reactions occur in vivo.
Chemiluminescence is the emission of light as the result of a chemical reaction. This is different to bioluminescence as the components of the chemical reaction do not necessarily occur in vivo.
Fluorescence is the emission of light by a substance that has absorbed radiation of a different wavelength. In most cases, absorption of light of a certain wavelength induces the emission of light with a longer wavelength. The fluorescent molecule is generally called a fluorophore and no chemical reaction is involved in the emission of light following excitation.
To take the example of bioluminescence, the use of organisms that have been genetically modified with genes encoding bioluminescent probes such as aequorin or various luciferases allows for the non-invasive visualization of many biological processes within living organisms and small animal models (1-3). Bioluminescence imaging is highly sensitive because signals can be optically detected within intact organisms, such as mice, due to the very low levels of intrinsic bioluminescence of mammalian tissues (4). Bioluminescent probes such as aequorin (5, 6) or the enzyme luciferase (bacteria, firefly, Cypridina (7) or Renilla (8)) have been incorporated into host cells or pathogens for bioluminescent imaging. These tools have been applied to studies ranging from infection and tumor development to calcium signaling. However, most of these biological markers emit light in the blue-green-yellow (400-650 nm) region of the color spectrum (9), which overlaps with the major absorption spectra of mammalian tissues and, in particular, with the absorption spectra of oxyhaemoglobin, de-oxyhaemoglobin and melanin, thus decreasing the efficacy of the detection (1, 10). Due to the presence of these absorbers, a major challenge in in vivo optical imaging is to achieve higher levels of sensitivity by developing optical probes emitting light in the red to near infrared (NIR) spectrum (650-900 nm), which is only weakly absorbed in living mammalian tissues.
Even with these drawbacks the use of this technology has readily applied itself to experiments involving models such as small rodents both anaesthetized and non-anaesthetized/freely moving (21). As indicated above in small rodent models (and more generally in mammals), the presence of various haemoglobins and tissues which readily absorb in the blue-yellow region of the color spectrum, reduces the ability to excite and detect fluorophores, or to monitor the luminescent signal produced by modified cells and bacteria if this is in this same spectrum.
Significant effort to improve the number of detectable photons from a bioluminescent source has led to the development of a vast array of new and/or improved bioluminescent probes and techniques that increase the number of emitted photons in the red region of the color spectrum (>650 nm). It has not proven too difficult to design fluorophores or fluorescent proteins that are both excited and emit in the red—near infrared (NIR) (22).
However, to achieve a similar red-shift for the bioluminescent emission has proven to be more difficult. One method towards establishing the desired red-shift has been to develop luciferase variants that are capable of emitting in this part of the red-NIR part of the light spectrum. However, most of the mutants/mutations developed so far have led to emissions in the yellow-green region (9) and there have been only a few examples of successful red bioluminescent production (11), and such red-NIR engineered proteins generally produce significantly less signal than the unengineered yellow-green versions.
Given the difficulty in producing red-NIR bioluminescence, significant effort has therefore been put towards creating applicable methodologies to produce a red-shift in the emitted photons using bioluminescence resonance energy transfer (BRET) (13). Several groups have already demonstrated the success of this technique utilizing luciferases bound to QDs (8) or other fluorophores (7) with large Stokes shifts.
In these previous methods, workers have investigated BRET by conjugating an eight-mutation variant of Renilla reniformis to a quantum dot, with the R. reniformis acting as the energy donor and quantum dots (QDs) as the energy acceptor thus achieving an emission peak at 655 nm from the QD following energy transfer (8). More recently, a biotinylated Cypridina luciferase was conjugated to a far-red fluorescent indocyanine derivative, providing an emission peak at 675 nm (7). However, in BRET, the donor and acceptor must be in close proximity (less than 10 nm (12-15)) as it involves a non-radiative energy transfer from the luminescent donor to the acceptor.
The use of BRET has been highly successful but can be difficult to implement since various synthetic techniques must be utilized in order to conjugate the donor luciferases to the acceptor QD or other fluorophore to achieve the desired resonance energy transfer. The need to bind the biological marker to a QD or other ‘red’ photon emitting acceptor, may make the biological marker less efficient as a means of visualizing ‘in vivo’ a cell, tissue or organism. This can be the result of interference with the function or ability of biological marker to produce a signal and/or move freely so as to associate with its target, where appropriate.